1) Background The work in my lab has revolved around the study of chondrogenesis and maintenance of the chondrocyte phenotype with attention focused on the changes that take place in cartilage leading to the arthritic diseases. We have sought to identify and analyze the factors that affect these aspects of cartilage biology. We have used both human chondrocytes and human mesenchymal stem cells along with retroviral gene transfer to explore the roles of secreted factors such as BMP2, Wnt3a and cartilage oligomeric matrix protein (COMP) in chondrogenesis in in vitro assays. We have also explored the roles of the intracellular histone deacetylases using these same cell systems. We have shown that these factors affect chondrogenesis in distinct ways. For example, BMP2 and COMP enhance chondrogeneis and chondrocyte survival while Wnt3a inhibits chondrogensis. These results provide us with the background to our present studies enabling us to fill gaps in our knowledge about the role of critical extracelllular and intracellular proteins in cartilage biology. 2) Objective of Present Studies Currently our objectives are three-fold: (1) The first objective is to define the role of COMP in chondrogenesis, chondrocyte survival and fibrosis. We will utilize both in vitro and in vivo systems for this objective. (2) The second objective is to determine the role of specific histone deacetylase enzymes in chondrogenesis, chondrocyte proliferation and the maintenance of the chondrocyte phenotype. Additionally we will initiate clinical studies to look at the ability of HDAC inhibitors to treat rheumatoid and osteoarthritis as well as chondrosarcomas and synovial sarcomas. Both in vitro and in vivo systems will be used for this objective. (3) Our third objective is to use genetic screens to identify genes whose expression enhances chondrocyte survivability. 3) Results During the Past Year The results of the past year are grouped according to the Objectives outlined above. (1) Analysis of the structure and function of the COMP Gene. Cartilage oligomeric matrix protein (COMP) is a specific component of cartilage, tendon and ligament. Mutations within the COMP gene lead to skeletal malformations and early onset arthritis. While COMP appears to play a crucial role in cartilage biology its exact role is unknown. We have analyzed the role of COMP in regulating chondrogenesis, adipogenesis and osteogenesis as well as in cell survival. We have utilized both human and mouse mesenchymal stem cells (MSCs) since these cells can differentiate in vitro into chondrocytes and osteoblasts. We have ectopically expressed COMP (wild-type and mutants) via plasmid and retroviral expression vectors and have assayed the resulting effects on chondrogenesis, adipogenesis and osteogenesis. We find that COMP enhances chondrogenesis, adipogenesis and osteogenesis in vitro. By expressing specific domains of COMP in these human MSCs we will be able to isolate and identify regions of COMP that specify its differentiation-enhancing functions. We have also assessed the role of COMP on cell proliferation and have found that specific domains of COMP have survivability functions. Future analysis will be directed at identifying the cellular receptors for COMP and determining its mode of action in terms of differentiation and proliferation. In addition, we will generate COMP transgenic mice. We will express wild-type and mutant COMP proteins in cartilage to determine the extent of any developmental defects in these mice and in particular those that affect cartilage. These approaches should help to shed light on the contribution of this important ECM molecule to cartilage biology and osteoarthritis. Additionally, we have been able to determine that COMP is not only a component of normal cartilage, tendon and ligament, but is a part of TGFbeta-initiated fibrosis in a variety of tissues. This includes skin, muscle, nerve etc. The data indicates that any tissue undergoing wound healing will likely express COMP. This could have important implications in the formation mechanical stability of fibrotic lesions, regardless of the tissue. (2) We have been able to identify the prominent HDAC enzymes in both primary human chondrocytes and mesenchymal stem cells. In addition we find that inhibition of one of these enzymes lowers the rate of chondrocyte cell proliferation. Further, when chondrocytes and MSCs are treated with HDAC inhibitors we find that this only mildly affects their viability and differentiation status, while these inhibitors have a profound effect on the growth of human chondrosarcoma and synovial sarcoma cells. (3) It is well recognized that there are multiple causes for the initiation of OA, some of which involve the effect of toxic factors directly on cartilage. In an attempt to identify genes that play critical roles in chondrocyte survival and chondrogenesis, we have initiated functional screens, using human cDNA libraries cloned into a retroviral expression vector. These retroviral cDNA libraries were used to infect chondrosarcoma cells, MSCs and chondrocytes. In our first screens we then have begun the process of selection for specific characteristics, such as for survival of cells treated with agents known to be toxic to bone and cartilage. For example, we have assayed for proliferation in media lacking growth factors or in the presence of toxic agents such as retinoic acid (RA), tumor necrosis factor (TNF), interleukin 1 (IL1) or the heavy metals lead (Pb) and gadolinium (Gd). Through this approach we have identified novel genes whose products that aid in the survival of these cells. The results of these initial screens are very promising for it has helped us identify a number of genes that one would not normally expect to be part of these processes. In the future we will use the information derived from these screens along with retroviral gene transfer approaches to express these potential chondro-protective genes in both cartilage and in MSCs. We will then determine the outcome of this expression on the survivability of chondrocytes in vitro and cartilage in vivo. 4) Conclusions and Significance With Future Directions In the last two to three decades, much progress has been made in the discovery of the causes and treatments of the various forms of arthritis. However, the initiating events for these diseases are not well understood and many of the treatment regimens are suboptimal at best. Since arthritis is such a debilitating disease that affects such a large proportion of the population, much effort should be made at uncovering the root cause and identifying treatment. I believe we have made excellent progress in defining new areas for exploration into cartilage biology and the treatment of cartilage diseases. We have explored the role of COMP, a prominent cartilage protein, in the role of cell death and survival and are attempting to determine its role in the long-term progression of disease. Further, we have explored the role of HDACs and their corresponding inhibitors in chondrogenesis. This is a completely untouched area that holds much promise for important discoveries in not only an understanding of the basic program of chondrocyte differentiation and development but in the treatment of cartilage diseases through the use of specific HDAC inhibitors. Finally, our use of a genetic screen to identify genes that block chondrocyte dedifferentiation may lead to the discovery of new genes that are involved in many aspects of cartilage biology.